Concentration and purification of interferons, viral inhibiting substances (vis), viral inhibiting factors (vif), or viral inhibitory material



June 14, 1966 LAMPSON 3,256,152

CONCENTRATION AND PURIFICATION OF INTERFERONS, mm. INHIBITING sussmucas(VIS), VIRAL INHIBITING FACTORS (VIF), on VIRAL INHIBITORY MATERIALFiled June 14, 1965 FLOW SHEET OF VIRUS INHIBITINGSUBSTANCE@NTERFERON)PURIFICATION Egg allantoic fluid 0 0.15N H0104, 5 C

centrifuged 30 min. at 1500 rpm Ppt. (discarded) Supernate pH adjustedto 7-8 0.02M Zn(Ac) 2 centrifuged 1 hr. at 1500 rpm Zinc. ppt. Supernate(discarded) Suspended in 0.2N HCl, 5 C pH 2.5 centrifuged 1 hr. at 2000rpm Supernate Ppt. (discarded) lg Dialyzed 24 hrs. against 0.9% saline 2pH adjusted to 7.0-7.2 3) 0.02M Zn(Ac) Zinc ppt. Supernatg (discarded) lDissolved in 0.2N HCl, 5 C. pH 2.5 2 Dialyzed 24 hrs. against 0.9%saline 3 Dialyzed 2 1 hrs. against 0.01M phosphate,

1) Centrifuged 1 hr. at 2000 rpm Ppt. (discarded) SupernateChromatography on CMC Column size 12" x 3/ 1" Stepwise NaCl gradient toPhosphate pH 6.0, followe Fraction eluted with 0.01M P04, pH 8,

l Dialyzed against 0.9% NaCl 3 pH adjusted to 7.2

4 Centrifuged 1 hr. at 2000 rpm Supernate (discarded) Zinc ppt.

1% Dissolved in 0.2N HCl, pH 3 0. 1M in 0.01M d by pH gradient 6-8 0.1MNaCl Dialyzed against 0.9% saline Dialyzed against 0.01M phosphate, pH oCentrifuged 1 hr. at 2000 rpm Supernate (8.5 m1.

Rechromatography on CMC Column size 2" x 1/2 Stepwise NaCl gradient to0. 1M in 0.01M phosphate, pH 6, followed by pH Fraction eluted with0.01M P0 pH 8, O-lM NaCl 1 Dialyzed against 0.9% NaCl 2 0.02M Zn(Ac)2 3pH adjusted to 7.

centrifuged 1 hr. at 2000 rpm.

Supernate (discarded) Zinc ppt.

' Dissolved in 0.2N HCl, pH 3 Ppt. (discarded) gradient 6-8 l 2 Dialyzedagainst 0.9%NaCl 3 centrifuged 1 hr. at 2000 rpm SupernatecontaininglNTERFERON (4m!) Ionophoretic or Zone electrophoreticseparation oi INTERFERON Ppt. (discarded) INVENTOR. GEORGE P. LAMPSONUnited States Patent s 256 152 CONCENTRATION AND PURIFICATION OF IN-TERFERONS, VIRAL INHIBITING SUBSTANCES vrs VIRAL INHIBITING FACTORS(VIF), OR

terms being synonymous. In this specification'and claims TABLE I.SUMMARYOF the term interferons shall be used and shall be understood to applyto the same active principle identified by the various terms employed inthe prior art. The invention more particularly is concerned with theconcentration VIRAL INHIBITORY MATERIAL 5 and purification ofinterterons from fluids containing these George P. Lampson, Hatfield,Pa., assignor t0 Merck & active substances,

' C0" y, a corporation of New Jersey The process of this invention can:be used to concen- Flled June 1965 N 463,907 trate and purifyinterferons which had been produced by 16 Clams (CL cells cultured invitro or by host cells in situ after their This application is aOOl'ltlIlllfltlOIl-lll-Pfil'tOlfnlY US. exposure to foreign nucleicacids Whether [from viral, aniapplications, Serial No. 384,749 filedJuly 23, 1964 (now mal or microbial sources but particularly frominfective abandoned), which is a continuation in-part-of applicaornoninfective viruses which generally induce the syntion, Serial No.244,222 filed December 12, 1962 (now thesis of larger quantities ofinterferon. Crude interabandoned), which in turn was acontinuation-impart feron-containing fluids can be harvested from suchtreated of application Serial No. 186,133 filed April 9, 1962 (now 5systems, and the interferons separated therefrom by the abandoned).method of this invention.

This invention is concerned with the concentration and Representativesystems known to induce the synthesis purification of viral inhibitingsubstances (VIS), which of interferon are identified in Table 1. Each ofthese are art recognized by the terms interferons, viral inhibitingsystems can be used to obtain the crude harvest fluid factors (VIP), andviral inhibitory material, all of said 0 from which the interferoncomponent can be concentrated and purified by the process of thisinvention.

INTERFERON SYSTEMS Interferon-Producing System Interferon-Testing SystemSource of Data Type of Virus Actrve or Method Cell, Tissue or Host Test;Cell, Tissue or Challenge Virus of lnactivatlon Host Yellow Fever 17 DDActive 011101! m ryo Chick embryo. Asibi; West Nile Lennett et al., J.Exp. High Strain. t Med. 83: 195 (1946).

Influenza Type A Heat ultraviolet rays. Ohlck chorloallantolcOhorioallantoic mem- MEL; PR5; Sendai; Isaacs et al., Proc. Roy.

(MEL, PR8). membrane, in vitro brane, in vitro; Newcastle disease; Soc,London, s.B. 147:

and in ovo. rabbit, intradermal. vaccinia. 258 (1957).

Isaac et al., Proc. Roy.

800., London, 5.3. 147: 268 (1957). N agano, Y., et al.,

Oompt. rend. Soc. de biol. 154: 2166 (1960). Burke et al., Brit. J.

Exper. Path. 39: 452 (1958). Ohorioallantoic mem- Vaocinia; cow-pox-Isaacs et al.', Brit. J. v brane, in ovo. Path. 39: 447

Influenza Type A Ultraviolet rays Monkeyidney C 1 Ohorioallantoic rnern-MEL Burke and Isaacs, Brit. (MEL)- Sendai. culture. brane, in vitro. 1Path. 39:

Fowl plague; Newcastle .....(10 Ch c ch o l fio c do Influenza Burke andIsaacs, Brit,

disease; influenza B membrane, in vltro. I. Exper. Path. ibid.

Lee

lngiueriza T e A Active do. -dodo- Burke and Isaacs, Brit.

I Y J'. Exper. Path. ibid.

V-n-pinia do Rabblt skin and Rabbit skin, in vivo Vaccinia Nagano andKojima,

' rabbit testes in v1vo. Oompt. rend. Soc. de.

biol. 152: 1627 (1958). Influenza Type A do Oali-k1dney ccll-cul-Calf-kidney cell 0111- Sendai Tyrell, Nature (London) ture;chorloallantoic ture; chorioallantoic 184 (Supp. 7): 452

membrane, 1n vltro. membrane, in vitro. (1959 Influenza Type AUltraviolet rays Rabbit-kidney cell Rabbit skin, in vivo. VacciniaIsaacs and Westwood, L), culture. Lancet 2: 324 (1959). Influenza Type A.....de Chorioallantrglc mem- Chick cell culture West Nile; Bunyam-Porterfield, Lancet 2;

(ME brane m vitro. wera; Western 326, (1959). Isaacs and equineencephalo- Westwood, Nature myelitis. (London) 184 (Supp. l 1): 1232(1959). Poliovirus Type 2 Active Human amnion; kid- Human amnion; kid-Polioviruses Type 1, Ho and Enders, Proc.

O) ney cell culture. ncy cell culture. 2 and 3; EOHO Nat. Acad. Sc. 385a Type 9; vaceinia; (1959).

herpes simplex; Idem. Virology 9: 446 Sin s. (1959). Influenza, Type Aws do Lntrallantoic, in ovo Chick-embryo cell Eastern equine en- Wagner,Bact. Rev. 24:

cul ure. cephalomyelitis. 151 (1960).

Newcastle disease; Ultraviolet rays MON cell culture MON; human kidney;Vesicular stomatitls; Henle et al., J. Exper. mumps; WS influenza;rabbit kidney; influenza Type A; Med. 525 (1959). S dai, moise-embryocell Sendal.

cu ure.

Mim'ms dn Mouse-fibroblast cell MON; mouse fibro- Vesicularstomatitis.-. Henle ct al., J. Exper.

culture. blast cell culture. Med. ibid.

MCN mumps 1 Active MON-mumps cell MON do Henle et al., J'.'Exper.

culture. Med. ibid.

H L -RMC HeLa-RMO cell Human-kidney and Poliovirus Type 1 Ho and Enders,Virology culture. amnion cell culture. 9: 446 (1959). Vesicularstomatitis... Chick-embryo cell Chick-embryo cell Vesicular stomat1t1sCooper and Bellett, J

culture. culture. gsgifvlicrobiol. 21: 485

Bellett and Cooper, J. Gen. Microbiol. 21: 498 H I 11 P 1 'r 2 141 11 EPoliovirus T e 1 2 Formalin Human-amnion cell umanamnion ce 0 iovirusype 1, ivisatos an dipidis d 3, YD culture. culture. and 3. Ztschr. f.Immunitatsforsch. u. exper.

Therap. 119: 344 (1960) TABLE I.-SUMMARY 0F INTERFERON SYSTEMS OQntinuedInterferon-Producing System Interferon-Testing System Source of DataType of Virus Active or Method Cell, Tissue or Host Test Cell, Tissue orChallenge Virus of Inactivation H051;

' Z T e 3 A ti KB cell culture Human-amnion cell Poliovirus Type 2Chany, Compt. rend. Pammfluen a yp culture. Acad. (1. so. 250: 3903(1960). Idem. Virology 13: 485 b 11 E 11 1 1 'dn't h k InfluenzaT 7 eAPR .do Mouse lung, in vivo-.. Mouseem ryo ce ncep a omyocarsaacs an 1 ccoo 3 p a) culturemice intraditis; polyorna; Lancet 2: 69 (1960).

pcritoneally. Bunyamwera. Hitchcock and Isaacs,

Brit, M. J. 2: 1268 (1960). Allison, Virology 15:

47 (1961). Mumps Ultraviolet rays HeLa-ccll culture HeLa-ccllculture"... Piliovirus Type 1. Cantell, Arch. f. d. ges.

XllSfOlSOh. 10: 510 Tick-borne encephalitis Active Chick-embryo cellDuck-embryo cell Western equine en- Vilcek, Nature (London) culture.culture. cephalomyelitls. 187: 73 (1960). Mumps Ultraviolet raysRabbit-kidney cell Rabbit eyes Vaccinia Cantell and Tommila, culture.Lancet 2: 682 (1960). Foot-and-mouth disease Active Calf-kidney cellFOOHmd-mouth Calf-kidney cell Dinter, Acta path. et

(Type 0). culture. disease (Type A5). culture. irlcgoglalggandinav.

: 7 ONyong-nyong do Adult mouse brain, Mouse fibroblast Encephalornyo-Hitchcock and Porterin vivo. carditis. gelbvirology 13: 363 Sindhi: doChick-embryo cell Chick-embryo cell Vesicular stomatitis Ho, Proc. Soc.Exper.

culture. culture. Biol and Med. 107:

639 (1961). Idem. J. Immunol.

(in press). Mayaro do "do d0 Sindbis Henderson and Taylor,

. Virology 13: 477

1961 do Human-amnion cell Human-amnion cell do De Maeyer and Enders,

culture. culture. Proc. Soc. Exper.

Biol. and Med. 107: 573 (1961). Influenza Type A Ultraviolet raysHuman-anion, rhesus Rabbit Skin and Vaccinia Andrews, Brit. M. J.

(MEL). and cynomolguscynomolgus mon- 1: 1728 (1961).

monkey-kidney, key skin in VlVO. chick-fibroblast and rabbit-kidney cellculture. Polyoma. Active Mouse-fibroblast cell Mouse-fibroblast cellEncephalomyo- Allison, Virology 15:

culture. culture. carditis. 47 (1961). Influenza- Ultraviolet raysChorioallantoic mem- Chick-embryo cell Chikungunya and Isaacs, Baron,Virology brane, in vitro. culture other Group A 14: 450 (1961); Idem.,

. arbor viruses NDV. Virology 14: 444 (1961). Influenza Type A ActiveHuman-adult- .Human-thyrold- ECHO Type II; Sutton and Tyrrcll,

(Kunz). thyroid-gland, gland and calf- Sendai. Brit. J. Exper. Path.

embryo-lung and kidney cell culture. 42: 99 (1961). embryo-kidney cellculture. West Nile do Adult mice, intra- L-cel cu tu e Mcngo Vainio etal, Virology cerebrally. 14: 385 (1961). Sendai do Hmnan-amnion cellHumamamnion cell Sindbis Gresser, Proc. soc.

culture. culture Exper. Biol. and Med.

108: 303 (1961). Do do Human leukocytes .do do Gresser, Proc. Soc.

Exper. Biol. and Med. 108: 799 (1961). Vaccinia Active and ultra-Mouse-embryo-cell A1 mouse-cell l1nes Vaccinia; Sindbis; Glasgow andHabel, J.

violet rays. lines (3B, ME 29) encephalornyo Exper. Med. 115: 503

and primary mousecardltis; herpes (1962). embryo cell culture. simplex:eastern equine encephalomyelitis; vesicular stomatitis. DA myxovirusHeat; ultraviolet rays. Monkey-kidney cell Monkey-kidney cell DAmyxovirus Hsiung, Proc. Soc.

culture. culture. Exper. Biol. and Med.

1 Interferon derived from persistently infected cell line.

It was discovered as a feature of this invention that interferonsprepared by the systems identified in Table I or by other systems knownto induce the synthesis of interferon can be separated in highconcentration from the crude harvest fluids thus obtained by firstadding a protein precipitating agent to remove virus and inactiveproteins from the interferon-containing fluid and thenprecipitating-interferon from the supernate by the addition of a heavymetal salt at a pH between about 6-8. A very substantial concentrationof interferon thus is obtained and the active material can be used inthis form as a test agent or as a viral inhibiting substance .for thepurposes hereinafter described.

It was also found as another feature of this invention that substantialpurification of interferon can be obtained by chromatographing the aboveobtained concentrated interferon on a carboxylic cation exchange resinand then eluting the active material by a continuous gradient orpreferably a stepwise gradient using an aqueous acetate or phosphatebuffer as the eluant, which gives up to about 2,000-fold concentrationof interferon with a purification factor of about 1700-fold dependingupon the ratio of impurities to active material. Further purificationand concentrationcan be efiected by again coprecipitating intenfcronwith a heavy metal salt as described above.

The high concentration and purification factors mentioned above (andalso to be referred to in the following discussion) are obtained whenthe starting fluid contains a very large amount of non-interferonmaterial, as is (suitable for small quantities or to detect the presenceof residual impurities) or by zone electrophoresis (especially suitablewhen larger quantities of highly concentrated and purifiedinterferon-containing materials are involved).

A crude harvest fluid containing interferon can be prepared by anywell-known method, such as any of the methods described in the papersidentified in Table I, and the active substance then can be concentratedand purified by the novel method of this invention. Two systemsgenerally are used to induce the elaboration of interferon, either bygrowing the interferon inducing agent in an embryonated egg or bygrowing the interferon inducing agent in tissue culture. Representativeexamples of these two systems are described below as illustrative ofthose methods suitable to prepare crude harvest fluids containing aninterferon.

An interferon-containing allantoic fluid can be prepared usingembryonated eggs, advantageously hens eggs, which have been incubatedfor 9 to 12 days. The eggs are infec-ted allantoically with live orinactivated virus especially RNA or DNA virus, advantageously byinjecting in about a 0.20 ml. inoculum dose between about to 10 butpreferably 10 EID (eggs infectious doses 50) of virus. Influenza virusis particularly suitable for the preparation of an interferon in thissystem.

The infected eggs then are incubated for at least 72 hours, butpreferably for 96 and at most 120 hours, at 36-37 C. after which theeggs are chilled and the allantoic fluid containing interferon iswithdrawn.

Other live or inactivated viruses can be used in place of influenzavirus mentioned above including especially measles virus, herpessimplex, vaccinia virus, Newcastle disease virus, encephalomyelitis,yellow fever and the like.

An interferon containing tissue culture filtrate (TCF) is prepared byinoculating the interferon inducing agent such as a live or inactivatedvirus, into tissue culture and incubating until maximum production ofinterferon has been induced. When using live organisms maximuminterferon production generally is reached at an advanced state of virusgrowth which can be determined by observing cytopathogenic changes inthe tissue culture cells. When using inactivated organisms, maximuminterferon production is determined by testing small samples of thetissue culture fluid. At the end of the interferon production period,the tissue culture filtrate is harvested by conventional methods.

The tissue cultures are prepared by well known methods using tissuesthatare conventionally employed for this purpose including mouse embryofibroblasts or lung tissue, chick embryo fibroblast cells orchorioallantoic membrane, monkey kidney tissue, hamster kidney orembryonic tissue, rabbit skin, testes or kidney,-calf kidney, L cells,human embryonic lung and kidney tissue, hum-an amnion and the like.

Live herpes simplex virus is particularly useful in the production of aninterferon when grown in tissue culture as described above. Other virusor interferon inducing agent can and have been used, however, for thispurpose including chickungunya, SV rubella, measles, mumps,parainfluenza virus (types 1, 2, 3), influenza viruses, polyoma,vaccinia, foot and mouth disease virus, eastern and westernencephalomyelitis virus, poliomyelitis virus, Newcastle disease virus,adenovirus, rickettsia, yellow fever and the like.

Interferon can be concentrated and purified from the crude harvestfluidobtained by the egg culture or tissue culture procedures of theprior art by the process of my invention by adding to theinterferon-containing fluid a protein precipitating agent, such asperchloric acid, trichloracetic acid, sulfosalicylic acid,tetrametaphosphate and the like to precipitate virus (for example, thevirus diaminetetraacetic acid tetra-sodium salt).

'tates, propionates, or chlorides.

protein precipitating agent is added to give a 'final concentration ofbetween 1 to 3%.

The interferon, which is stable at low pH, remains in the supernate andcan be precipitated therefrom by adjusting the pH to between 6-8 butpreferably between 7-8- with a strong base, advantageously sodium orpotassium hydroxide, and then adding a solution of a heavy metal salt toa concentration of between about 5 to millimolar, advantageously between10 to 20 millimolar. Heavy metal salts which are especially useful inprecipitating the interferon are cobalt, zinc, calicum, mercury,strontium, barium' and the like halides of salts of an organic acid, andin particular the cobalt, zinc, calcium, mercury, strontium or bariumacetates, glycinates, lac- For all practical purposes, zinc acetate canbe used as it is readily available and quite suitable as a precipitantfor interferons.

The heavy metal salt precipitate containing the interferon is removedpreferably by centrifugation or other mechanical means. The pelletobtained then is stirred at 0-5 C. and treated either with sufficientstrong acid to obtain a pH of about 2-3 to selectively solubilize theinterferon or with a chelating agent to selectively bind the heavy metalsalt while the interferon remains in solution.

Suitable agents are strong mineral or organic acids as perchloric acid,hydrochloric acid, trichloracetic acid and the like or chelating agentssuch as Versene (i.e., ethylene- Hydrochloric acid (0.2 N) can be usedwith advantage in this step. The suspension obtained then is clarifiedby centrifuging and the supernate which contains the heavy metal salt insolution as well as the interferon is dialyzed against saline to removeresidual heavy metal salt.

The dialyzed solution thus obtained (referred to hereinafter as dialyzedsolution I) contains about a 10- to 20-fold concentration of theinterferon when employing allantoic fluid-containing-interferon as thestarting fluid.

The interferon in dialyzed solution I, if desired, can be furtherconcentrated by repeating the above described precipitation with a heavymetal salt followed by dialysis to provide a 100-fold concentration ofthe interferon with a 10- to 20-fold purification in dialyzed solutionII.

Dialyzed solutions I or II contain a useful concentration of interferon.It can be used, for example, to prevent Newcastle disease virusinfection as will be shown hereinafter, or to inhibit tumor formation byRous Sarcoma virus in the wing web of l-day old chicks.

inoculant) and a large amount of inactive protein. The a The interferonin either dialyzed solutions I or II can be purified further bychromatographing on a carboxylic cation exchange resin by a continuousgradient or preferably a stepwise gradient using aqueous acetate orphosphate buffer as the eluant. Further concentration and purification,if desired, can be obtained by again precipitating the interferon fromthe eluant by use of a heavy metal salt as described above. Apurification factor up to about 2000-fold can thus be attained.

An adsorption column is prepared by passing an aqueous buffer such asacetate or phosphate buffer solution having a pH of about 5.8 to 6.4through a column of adsorbent until the eflluent has a pH of about 5.8to 6.4. A solution of interferon which can have activity of up to about25,000 units per ml. and which has been dialyzed against a buffer havinga pH of 5.8 to 6.4 and an ionic strength of 0.03-0.08 is added to acolumn of carboxy methyl cellulose or other suitable weak cationicexchangers such as a carboxylic cation exchangeresin. After the additionis complete, additional buffer solution having a pH of 5.8 to 6.4 ispassed through the column which removes a considerable amount of proteinhaving no interferon activity.

The concentration of protein in the efliuent is measured by the Lowrycolorimetric method as described in J. Biol Chem. 193: 265-75 (1951), O.W. Lowry et al., Protein Measurement with Folin Phenol Reagent.

An additional amount of proteinaceous material having no interferonactivity is removed from the adsorbent by slowly passing a buffersolution having a pH of 5.8 to 6.4 with a stepwise increase in ionicstrength to about 0.13 through the column until the effluent issubstantially protein free. This solution is prepared from a buffersolution having a pH of about 5.8 to 6.4 by the addition of an inorganicsalt, preferably sodium chloride.

Substantially purified interferon is removed from the adsorbent bypassing through the columnabuffer solution having a pH of about 7.4 to8.8 and an ionic strength of about 0.13 to 0.18.

The resin employed for adsorbing the interferon preferably is acarboxylic cation exchange resin and for the purposes of this inventionthe resin can be any of the carboxylic type resins which are available.It was found that carboxymethylcellulose (CMC) prepared by the methoddescribed by Peterson and Sober, J. Am. Chem. Soc. 78, 756 (1956) ishighly effective in providing efficient adsorption and release of theinterferon. Other suitable carboxylic cation exchange resins includeAmberlite IRC-SO and Amberlite XE-54, both obtainable from Rohm and HaasCompany, are Permutit H-70, sold by the Permutit Company, or otheravailable carboxylic cation exchange resins.

The concentrated and highly purified interferon obtained is effective aswill be shown below, in interfering with the multiplication of Newcastledisease virus, and is therefore potentially useful in the prevention ofNewcastle disease in fowl. In addition it is effective in interferingwith the ability of the Rous Sarcoma virus in producing tumor growth orfor the purposes described in the papers identified in Table I.

The major portion of residual impurities, if any, in theinterferon-containing solution obtained as described above can beremoved either by ionophoresis or zone electrophoresis.

The interferon-containing solution obtained following chromatography isfurther purified by ionophoresis by use of cellulose acetate paper orany other type of paper having a low adsorptive capacity for theinterferon. The interferon-containing solution is applied in a narrowzone on cellulose acetate paper or other suitable paper with the endsimmersed in electrode vessels containing any buffer solution in the pHrange of 4-10 but preferably borate buffer of pH 8.9. An electriccurrent of 0.4 milliampere/cm. (ma/cm.) width of the paper is appliedfor from 1 to 3 hours. The paper then is cut into narrow strips and theinterferon extracted with 0.9% saline solution containing 0.05 N aceticacid or 0.9% saline containing bovine serum albumin and Tween 80.

Alternatively, larger quantities of chromatographed interferon can bepurified by zone electrophoresis on a bed or column of Pevikon C-870 (acopolymer of polyvinyl acetate and polyvinyl chloride, obtainable fromStockholm Superfostat Fabrika A.-B., Stockholm, Sweden), powderedcellulose acetate paper, or any other media having a low adsorptivecapacity for the interferon. A concentrated solution containinginterferon is applied in a slit made in a preformed bed of Pevikon C-870or other suitable media with the ends connected through means of filterpaper to electrode vessels containing any suitable bufler in the pHrange of 4 to but preferably borate buffer, pH 8.9. Five to ten voltsper cm. length of bed is applied for from 6 to 18 hours at 0 to C. butpreferably maintaining the temperature at 0-5 C. The bed is then slicedinto narrow segments which are extracted with 0.9% saline solutioncontaining 0.05 N acetic acid or 0.9% saline solution containing bovineserum albumin and Tween 80.

The flowsheet forming part of this application which illustrates atypical run in which an interferon is concentrated and purified from eggallantoic fluid, will be described in more detail in the followingexample. The same procedure is used in whole or in part to concentrateinterferon from tissue culture filtrate or from any of the systemsdescribed in the papers identified in Table I. As

mentioned above, it is not necessary to carry out all of the stepsdescribed in the examples or in the flowsheet to obtain a highlyconcentrated and purified interferon that can be used to inhibit virusreplication, as the interferon obtained by treating the crude harvestfluid with the protein precipitating agents mentioned above and thencoprecipitating the interferon from the supernate with a heavy metalsalt by the procedure described above yields a useful concentration ofthe active principal.

EXAMPLE 1 Concentration and purification of interferon from allantoicfluid Step 1.To 50 liters of egg allantoic fluid harvest containing 256units of interferon activity per ml. [see footnote 1, appended to TablesII and III for definition of unit] were added 5.5 liters of 1.5 Mperohloric acid with rapid stirring at 5 C. After standing 30 minutes,the mixture was centrifuged 30 minutes at 1500 r.p.m. in a refrigeratedcentrifuge.

This step can be carried out using trichloroacetic acid, sulfosalicylicacid or tetrametaphosphate in place of perchloric acid to obtain similarresults.

The allantoic fluid used in this step was prepared by injectingallantoically 10 EID of influenza A virus, WS strain, E 5372 (obtainablefrom the Rockefeller Institute, New York 21, New York, or from Merck andCo.,

Inc., West Point, Pennsylvania), in a 0.20 ml. inoculum dose into each9- to 10-day old embryonated hens eggs, incubating 96 hours at 36-37"C., after which the eggs are chilled and the allantoic fluid containinginterferon withdrawn.

Other active or inactive viruses or other agents known to induce theelaboration of interferons in embryonated eggs can be used in place ofthe particular influenza virus strain mentioned, such as other strainsof influenza A, B or C virus, as the PR strain of influenza A virus,Melbourne strain of influenza A virus and the like or measles, herpessimplex, vaccinia, Newcastle disease viruses and the like to induce thesynthesis of interferon in the allantoic fluid.

Step 2.-The supernate (55 liters containing 256 units of interferonactivity per ml.) was adjusted to pH 7-8 with sodium hydroxide and 0.4molar zinc acetate was added with stirring to a final concentration of20 millimolar. After standing overnight at 5 C. as much super nate wasaspirated off as was possible and the remaining zinc precipitate sludgewas centrifuged one hour at 1500 r.p.m. in a refrigerated centrifuge.The collected precipitate containing zinc acetate and interferonwas'stirred with sufficient cold (0-5 C.) 0.2 N HCl to lower the pH to2-3 to dissolve the precipitate.

The precipitate also can be dissolved using other strong mineral ororganic acids or by employing a chelating agent, as Versene.

The solution was clarified by centrifugation at 1500 r.p.m. for one hourin a refrigerated centrifuge. The interferon-containing solutionobtained which had been concentrated 20-fold to 2.6 liters, was dialyzedovernight against 10-20 volume of 0.9%v sodium chloride to remove thezinc ions and to give a solution containing 3,840 units of interferonactivity per ml.

Step 3.The 20-fold, dialyzed concentrate was adjusted to a pH of 7.0-7.2with sodium hydroxide and 0.4 molar zinc acetate was added with stirringto a concentration of 20 millimolar. After standing 3 hours or more at 5C., the precipitate was centrifuged one hour at 2,000 r.p.m. Thesupernate was discarded and the precipitate was dissolved in sufiicientcold (0-5" C.) 0.2 N HCl to obtain a pH of 2-3, and then dialyzedovernight against 50-100 volumes of 0.9% sodium chloride to remove thezinc ions. The interferon containing solution (490 ml.) was concentratedto 100-fold with a 10- to 20-fold purifi cation by this procedure, andhad 12,800 units of activity per ml.

Similar results are obtainable using any of the aforementioned heavymetal chlorides or heavy metal salts of organic acids especially thecobalt, zinc, calcium, mercury, strontium or barium acetates,glycinates, lactates, propionates or chlorides.

Step 4.The interferon in the solution obtained in Step 3 was furtherpurified by chromatographing on carboXymethyl-cellulose using a stepwisesodium chloride and pH gradient in 0.01 M phosphate buffer as follows:

(a) The 100-fold concentrated interferon (490 ml.) from Step 3 wasdialyzed overnight against 30 volumes of 0.01 M sodium phosphate, pH 6.Y

(b) The dialyzed interferon containing solution was applied to a columnof carboxymethylcellulose, 12" X /1", which had been equilibrated With0.01 M sodium phosphate, pH 6. i

(c) Chromatography was carried out using a stepwise addition to thecolumn of the following buffers:

Buffer: Volume, ml.

0.01 M phosphate, pH 6 250 0.01 M phosphate, pH 6, 0.05 M NaCl 500 0.01M phosphate, pH 6, 0.10 M NaCl 2500 0.01 M phosphate, .pH 8, 0.10 M NaCl500 (d) The interferon was eluted with 0.01 M phosphate, pH 8,containing 0.10 M NaCl. The main peak of activity (19,200 units/ml.) wascontained in a fraction volume of 120 ml. The other fractions can berechromatographed in order to concentrate and to purify further theinterferon in each of them and to increase the yield.

Step .In order to carry out a second chromatography, the above 120 ml.fraction of interferon'was concentrated to 8.5 ml. by zinc acetateprecipitation as described in Step 3, to give an interferon-containingsolution having 153,600 units of activity per ml.

Step 6.The zinc acetate-concentrated fraction of inter- 10Buffer-Continued Volume, ml. 0.01 M phosphate, pH 6, 0.07 M NaCl 10 0.01M phosphate, pH 6, 0.08 M NaCl 10 0.01 M phosphate, pH 6, 0.10 M NaCl0.01 M phosphate,'pH 8, 0.10 M NaCl 50 The interferon was eluted with0.01 M phosphate, pH 8, containing 0.1 M NaCl, the main peak of activity(76,- 800'units/ml.) was contained in a fraction volume of 13.5 ml. Theother fractions can be rechroniatographed in order to furtherconcentrate and purify the interferon in each of them and to increasethe yield.

The chromatography described in Steps 4 and 5 can be carried out withother carboxylic cation exchange resins, such as Amberlite IRC-SO,Amberlite XE-54, Permutit H- or other available carboxylic cationexchange resins to obtain equivalent results in the furtherconcentration and purification of interferon.

Step 7.The active fraction from Step 6 was further concentrated to 4 ml.by zinc precipitation and dialysis as described in Step 3 .to give225,000 units of interferon activity per ml.

Step 8.Interferon was separated from the major portion of residualimpurity remaining following Step 7 by pipetting a 0.4 ml. aliquotthereof into a narrow slit made in a 17 cm. by 2 cm. Pevikon C-870block. One end of the Pevikon block was attached, through filter paper,to a positive electrode and the opposite end of the block was attached,through filter paper, to a negative electrode; each electrode beingimmersed in 0.03 M sodium borate buffer, adjusted to pH 8.9 withhydrochloric acid. A current of 3 milliamperes (150-170 volts) waspassed through the Pevikon block for 7 hours. At the end of the run, thePevikon block was sliced into 1 cm. or 0.5 cm. sections as indicated inthe following table and each section was separately extracted with asolution containing 0.05 N acetic acid and 0.9% sodium chloride and theextracts separately analyzed for protein and interferon activity. Thedata obtained-is given in the following table, wherein the area of thePevikon block sections are identified; the areas having been sliced fromthe end of the block attached to the anode,.and the areas having beensliced from the end of the block attached to the cathode. The first twopositive slices and the last two negative slices were each of 1 cm., theremaining slices being 0.5 cm. each.

TAB LE II Fonfootnotes see end of Table III.

Buffer: Volume, ml. 0.01 M phosphate, pH 6 10 0.01 M phosphate, pH 6,0.05 M NaCl 10 The degree of concentration and purification ofinterferon obtained by the various steps of the above pro- 0.01 Mphosphate, pH 6, 0.06 M NaCl 10 cedure is summarized in the followingtable:

TABLE III Interferon Protein, Sp Act., Percent PurificationFractionation Step Volume Activity, mgJml. units/mg. Recovery Factor 4units/ml. protein Crude Harvest 50 liters 256 5 0 52 1. Supernate fromperchloric acid 55 liters 256 2 2 116 100 2.

treatment of crude harvest. 2. First zine precipitation 3, 840 7. 4 52075 10 3. Second zinc precipitation 12, 800 15.6 820 50 16 4. Firstchromatography on CD1 19, 200 0.38 50, 500 18 970 5. Concentration ofactive fraction 153, 600 2. 7 57, 000 10. 2 1,100

from column with zinc acetate. 6. Second chromatography on CMC c. 76,800 0 85 92,000 8 1, 750 7. Concentration of active fraction 225, 000 238 95, 000 7. 2 1, 830

with zinc. 8. Final purification on Pevikon C- 2, 560 .0108 236, 000 4,000

1 The unit of activity reported in these tables is assayed in terms ofinterference with a Group A Arbo Virus such as Sindbis virus or easternequine encephalomyelitis (EEE) virus in monolayer cell cultures of thesame tissue used when synthesizing the interferon. Serial dilutions ofinterferon-containing fluid, 1 ml., is added to the cell eultures andincubated at 37 C. for 6 hours. After removing the fluid, each treatedcell culture is challenged \vlth 10f) TCIDm (100 tissue cultureinfectious doses) of EEE, Sindbrs or other Group A Arbo Virus. The unitof fluid which effects total prevention of eytopathology 1n the cellculture.

interferon is expressed as the reciprocal of the highest dilution ofinterferon-containing 2 Determined by the method described by O. H.Lowry et al, J. Biol. Chem. 193: 26575 (1951), Protein Measurement withFolin Phenol Reagent.

its ism =Pur1fieation factor.

The interferon concentrated by the above procedure, Steps 1 through 7,was tested for its effect on Newcastle disease virus (NDV) in one-dayold chicks and was found to protect some of the chicks from death causedby the virus and additionally to prolong the survival time of thosechicks not fully protected by the interferon. studies were carried outby two methods; one wherein the interferon was administered in two doses24 and 6 hours before the chicks were challenged and secondly whereinthe interferon was administered in two doses 24 and 6 hours before thechicks were challenged and again daily starting 18-20 hours after virusinfection and continuing for the number of times specified in thefollowing table. The designated units of interferon were administeredintraperitoneally as a 0.5 ml. inoculant. Newcastle disease virus alsowas administered intraperitoncally as a 0.5 ml. inoculant containing 5LD For comparison the crude harvest, i.e., the interferon-containingallantoic fluid (Step 1) used as the starting material in the novelprocedure of this invention, was tested along with the concentratedinterferon preparation obtained by the second zinc acetate precipitationstep (Step 4). Control birds which received only the challenge dose of 5LD doses of NDV in a 0.5 ml. inoculant also were included. The resultsof this study are reported in the following table.

The

perchloric acid and employing the crude harvest fluids containing aninterferon obtained by any of the methods described in the papersidentified in Table I or by other known methods to obtain similarresults.

The interferon-containing tissue culture filtrate (TCF) employed in thisstep was prepared by growing live herpes simplex virus upon tissuecultures of embryonic chick fibroblasts. The tissue culture was preparedfrom trypsinized chick embryo tissue from 9- to 10-day-old chickembryos, suitably by the method of Dulbecco and Vogt, J. ExperimentalMedicine, 99 (1954) 167. The trypsinized tissues were grown for 72 hoursin Blake bottles at around 36 C. in a suitable fluid growth medium suchas Medium 199 containing 1% calf serum (Proc. Soc. Exp. Biol. and Med.,73 (1950) 1). Live herpes simplex virus is inoculated in to fresh growthMedium 199 containing a suitable antibiotic as penicillin, streptomycinor a combination thereof and sufficient inoculum is added to each Blakebottle to give a final volume of 100 ml. of medium containing 3-10 TCID(tissue culture in fectious doses 50) of virus per ml. The Blake bottlesthen were incubated at 3637 C. for about 96 hours or until an advancedstate of virus growth is reached whichcan be determined by observingcytopathogenic changes TABLE IV.MEASUREMENT OF EFFECT OF ADMINISTRATIONOF CRUDE AND CONCENTRATED INTERFERON ON NEWCASTLE DISEASE VIRUS INONE-DAY OLD CHICKS Regimen of Treatment with Interferon FindingsSurvival Indices N0. animals Total surviving] Percent Average ExcessExcess Interferon fraction Pre-virus Post-virus units Tot su v valSurvival survival survival animals in days in days 2 X 64 U 1 128 3/1030 9. 9 23. 3 2. 2 2 x 64 U 448 4/15 27 10.0 20. 3 2. 3 2): 3,200 U 6,400 6/10 13.8 53.3 6.1 2 x 3,200 U 72, 400 5/ 33 10.5 26. 3 2. 8 2/306.7 7.7

1 Two doses each containing 64 units interferon.

EXAMPLE 2 Concentration and purification of interferon from tissueculture fluids (from chick fibroblast culture) Step 1.To 800 ml. oftissue culture fluid containing 48 units of interferon .activity per ml.were added 65 ml. of 2 M perchloric acid slowly with stirring at 5 C.After stirring l020 minutes, the mixture was centrifuged 30 minutes at1500 r.p.m. in a refrigerated centrifuge.

This step can be carried out using trichloracetic acid, sulfosalicylicacid or tctrametaphosphate in place of in the tissue culture cells.

to a final concentration of 10 millimolar.

foot and mouth disease, eastern and Western equine encephalomyelitis,Newcastle, polio and the like agents as used in the interferon-inducingsystems described in the papers identified in Table I to induce thesynthesis of an interferon in the desired tissue culture system.

Step 2.The supernate (850 ml. containing 48 units of interferon activityper ml.) was adjusted to pH 7 with sodium hydroxide and 0.4 molar zincacetate was added The pH was carefully adjusted to 7.4 by the additionof 2 N sodium hydroxide. After standing overnight at 5 C., as muchsupernate was aspirated off as was possible and the remaining zincprecipitate sludge was centrifuged 30 minutes at 2000 rpm. in arefrigerated centrifuge. The collected Zinc precipitate containinginterferon Was stirred with sufficient cold (5 C.) 0.2 N HCl to dissolvethe precipitate at a pH of 2-3. The interferon-containing solutionobtained which had been concentrated 13 fold to 60 ml., was dialyzedovernight against 50 volumes of 0.9% sodium chloride to remove zinc ionsand to give a solution containing 512 units of interferon activity perml.

Other heavy metal salts can be used in place of the zinc acetate,including the cobalt, zinc, calcium, mercury, strontium or bariumacetates, glycinates, lactates, propionates or chlorides, tocoprecipitate the interferon.

Step 3.To the dialyzed 13 fol-d concentrate was added 0.4 M zinc acetateto a concentration of 20 millimolar. The pH was carefully adjusted to7.4 with 2 N sodium hydroxide. C., the second zinc precipitate wascentrifuged one hour at 2000 rpm. The collected precipitate wasdissolved in sufiicient cold (05 C.) 0.2 N HCl to obtain a pH of 2-3 andthe solution then dialyzed overnight against 50-100 volumes of 0.9%sodium chloride to re- 35 After standing 3 hours or more at 5 Interferonfrom Allantoic Fluid (Example 1):

(1) Stable at pH 2-10 (2) Stable at 56 C. for 1 hour (3) Elutes fromCM-cellulose at pH 8 (4) Molecular weight 34,000 (5) Migrates towardanode at pH 8.9 on cellulose acetate paper 6) Destroyed by trypsinInterferon from Tissue Culture Fluid (Example 2):

(1) Stable at pH 2-10 (2) Stable at 60 C. for 1 hour (3) Elutes fromCM-cellulose at pH 8 (4) Molecular weight 36,000 (5) Migrates towardanode at pH 8.9 on cellulose acetate paper (6) Destroyed by trypsin TheCMC used for chromatography in Step 4 of Example 2 can be replaced byother carboxylic cation exchange resins, such as Amberlite IRC-50,Amberlite XE-54, Permutit H-70 or other available carboxylic cationexchange resins to obtain equivalent results in concentrating andpurifying interferon.

The degree of concentration and purification of interferon obtained bythe various steps of Example 2 is summarized in the following table.

TABLE V.-PURIFICATION OF INTERFERON FROM TISSUE CULTURE FLUID InterferonMg. Protein Sp. Act, Percent Purification Fractionation Step VolumeActivity, per ml. units/mg. Recovery Factor units/ml. Protein 1. CrudeHarvest--. 800ml 48 0.77 62 2. First zinc precipi- 60 m1 512 4. 2 122 802. 0

tation.

3. Sargon zine precip- 12.51111 2, 048 13. 9 147 67 2.4

4. Chromatography 10 512 0. 11

on CMC- 4, 660 13 75 Assays employed are those identified in Table IIand III footnotes. move zinc ions. The thus obtained interferon solutionEXAMPLE 3 (12.5 ml.) represents a 64 fold concentrate containing 2,048units of activity per ml.

Step 4.The above interferon concentrate was purified by chromatographyon carboxymethyl cellulose using a stepwise sodium chloride and pHgradient in 0.01 M phosphate buffer as follows:

(a) The 64 fold concentrated interferon 12.5 ml.) from Step 3 wasdialyzed overnight against 100 volumes of 0.01 M sodium phosphate, pH 6.

(b) The dialyzed interferon solution was applied to a column ofcarboxymethyl cellulose, 5" x /2", which had been equilibrated with 0.01M sodium phosphate pH 6.

(c) Chromatography was carried out using a stepwise addition to thecolumn of the following buffers:

Buffer: Volume ml.

0.01 M phosphate, pH 6 50 0.01 M phosphate, pH 6, 0.05 M NaCl 20 0.01 Mphosphate, pH 6, 0.10 M NaCl 90 0.01 M phosphate, pH 8, 0.10 M NaCl 50(d) The interferon was eluted with 0.01 M phosphate, pH 8, containing0.1 M NaCl. The main peak of activity (512 units/ml.) was contained in afraction volume of 10 ml. The other fractions can be rechromatographedin order to concentrate and purify further the interferon in each ofthem and to increase the yield.

Concentration of interferon from tissue culture fluids (from mouseembryo culture) Step 1.-To 500 ml. of crude tissue culture fluids con-55 taining 32 units of interferon activity per ml. were added 125 ml. of2 M perchloric acid. The mixture was stirred 20 minutes at 5 C. and thencentrifuged 30 minutes at 1500 r.p.m. in a refrigerated centrifuge.

This step can be carried out using other protein precipitating agentssuch as trichloracetic acid, sulfosalicylic acid, tetrametaphosphate andthe like to obtain similar results.

The interferon-containing TCF employed in this step was prepared bygrowing influenza A virus in mouse embryo tissue culture. The tissueculture was prepared from trypsinized, mouse embryo tissue from 15-18day old mouse embryos, suitably by the method of Dulbecco and Vogt,supra. Twenty Blake bottles containing the tissue culture were eachseeded with suflicient virus to provide a final volume of 100 ml. ofMedium 199 containing 3-10 TCID of Influenza A virus per ml. The Blakebottles then were incubated at 37 C. for 72 hours. At the end of thisperiod the TCP overlay was harvested by decantation and found to contain32 units of inter- 75 feron activity per ml. when assayed against EEEvirus refrigerated centrifuge.

' cedure identified in the footnotes to Tables II and III.

Step 2.The supernate from Step 1 (600 m1. containing the interferonactivity) was adjusted to pH 7 with 10% sodium hydroxide and 0.2 M zincacetate was added to a final concentration of 20 millimolar. The pH wasadjusted to 6.8 by the addition of 0.5 N sodium hydroxide and themixture wasallowed to stand overnight at 5 C.

Step 3.The precipitate was collected by centrifugation and dissolved in4 ml. of cold (5 C.) 0.2-N hydrochloric acid. The acid solution (pH 3)was dialyzed overnight against 100 volumes of 0.9% sodium chloride andfurther dialyzed against 100 volumes phosphate buffered saline pH 7 togive 5 ml. of solution containing 1,537 units of interferon activity perml. representing a 100 fold concentration of the active material.

EXAMPLE 4 Concentration of interferon from tissue culture fluids (fromrabbit kidney culture) To 500 ml. of crude harvest fluid containinginterferon obtained by the method described by Isaacs et al., Lancet 2:324 (1959) by inoculating rabbit-kidney cell cultures with InfluenzaType A(MEL) inactivated by irradiation with ultraviolet rays, is added125 ml. of trichlor- 16 sodium hydrochloric acid. The mixture is stirredabout /2 hour at C. and then centrifuged for /2 hour at 1500 r.p.m. in arefrigerated centrifuge. The supernate is separated and adjusted to pH 7with 10% sodium hydroxide and 0.2 M barium chloride is added to a finalconcentration of about 10 millimolar. The pH again is adjusted to 77.5by addition of 5% sodium hydroxide and the mixture allowed to standovernight at 5 C. The precipitate is collected by centrifugation anddissolved in 5 ml. of cold (05 C.) perchloric acid (final pH 2-3). Theacid solution is dialyzed overnight against 100 volumes of 0.9% sodiumchloride and further dialyzed against 100 volumes phosphate bufferedsaline (pH 7) to acetic acid. The mixture is stirred about /2 hour at 15C. and then centrifuged for /2 hour at 1500 r.p.m. in a The supernate isseparated and adjusted to pH 7.5 with 10% sodium hydroxide and 0.2 Mzinc acetate is added to a final concentration of about 10 millimolar.The pH is again adjusted to 7.0-7.5 by addition of 5% sodium hydroxideand the mixture allowed to stand overnight at 15 C. The precipiate iscollected by centrifugation and dissolved in 5 ml. of cold (05 C.) 0.2 Nhydrochloric acid (final pH 2-3). The acid solution is dialyzedovernight against 100 volumes of 0.9% sodium chloride and furtherdialyzed against 100 volumes phosphate buffered saline (pH 7) to giveabout 6 ml. of solution containing highly concentrated (about 100 fold)interferon.

EXAMPLE 5 Concentration of interferon from tissue culture fluids (fromhuman amnion culture) To 250 ml. of crude harvest fluid containinginterferon obtained by the method described by Ho and Enders,

' Proc. Nat. Acad. Sc. 385 1959) by inoculating human amnion cellculture with polio virus type 2 (RMC) is added 25 ml. of 10%sulfosalicylic acid. The mixture is stirred about /2 hour at 5 C. andthen centrifuged for /2 hour at 1500 r.p.m. in a refrigeratedcentrifuge. The supernate is separated and adjusted to pH 7 with 10%sodium hydroxide and 0.2 M calcium proprionate is added to a finalconcentration of about 25 millimolar. The pH again is adjusted to 7-7.5by addition of 5% sodium hydroxide and the mixture allowed to standovernight at 5 C. The precipitate is collected by centrifugation anddissolved in 2-3 ml. of cold (05 C.) 0.2 M hydrochloric acid (final pH2-3). The acid solution is dialyzed overnight against 100 volumes of0.9% sodium chloride and further dialyzed against 100 volumes phosphatebuffered saline (pH 7) to give about 2-3 ml. of solution containinghighly concentrated (about 40 fold) interferon. EXAMPLE 6 Concentrationof interferon from tissue culture fluids (from mouse fibroblast culture)To 500 ml. of crude harvest fluid containing interferon obtained by themethod described by Henle et al., J.

sodium tetrametaphosphate and adjusted to pH 2 with give about 5 ml. ofsolution containing highly concentrated (about 50 fold) interferon.

EXAMPLE 7 Concentration of interferon from tissue culture fluids (fromchick embryo fibroblast culture) To 500 ml. of crude harvest fluidcontaining interferon obtained by the method described by Vileck, Nature(London) 187: 73 (1960) by inoculating chick embryo fibroblast cellswith tick-borne encephalitis virus is added 26 ml. of 2 M perchloricacid. The mixture is stirred about /2 hour at 5 C. and then contrifugedfor /2 hour at 1500 r.p.m. in a refrigerated centrifuge. The supernateis separated and adjusted to pH 7 with 10% hydroxide and 0.2 M calciumglycinate is added to a final con- EXAMPLE 8 Concentration of interferonfrom tissue culture fluids (from human amnion cell culture) To 500 ml.of crude harvest fluid containing interferon obtained by the methoddescribed by DeMaeyer et al., Proc. Soc. Exper. Biol. and Med., 107:573(1961) by inoculating human amnion cell cultures with measles virus isadded 50 ml. of 10% trichloracetic acid. The mixture is stirred about /2hour at 3 C. and then centrifuged for /2 hour at 1500 r.p.m. in arefrigerated centrifuge.- The supernate is separated and adjusted to pH7 with 10% sodium hydroxide and 0.2 M strontium chloride is added to afinal concentration of about 10 millimolar. The pH again is adjusted to77.5 by addition of 5% sodium hydroxide and the mixture allowed to standovernight at 5 C. The precipitate is collected by centrifugation anddissolved in 5 ml. of cold (05 C.) 0.25 M Versene(ethylenediaminetetraacetic acid tetrasodium salt) (adjusted to pH 23).The solution is dialyzed overnight against volumes of 0.9% sodiumchloride and further dialyzed against 100 volumes phosphate bufferedsaline (pH 7) to give about 5 ml. of solution containing highlyconcentrated (about 50 fold) interferon.

EXAMPLE 9 Concentration of Interferon from Tissue Culture Fluids (frommouse fibroblast cell culture) To 500 ml. of crude harvest fluidcontaining interferon obtained by the method described by Allison,Virology 15: 47 1961), by inoculating mouse fibroblast cell cultureswith polyoma virus is added 26 ml. of 2 M perchloric acid. The mixtureis stirred about /2 hour at 5 C. and then centrifuged for /2 hour at1500 r.p.m. in a refrigerated centrifuge. The supernate is separated andadjusted to pH 7 with 10% sodium hydroxide and 0.2 M zinc acetate isadded to a final concentration of about 20 millimolar. The pH again isadjusted to 77.5 by addition of 5% sodium hydroxide and the mixtureallowed to stand overnight at 5 C. The precipitate is collected bycentrifugation and dissolved in 5 ml. of cold (5 C.) hydrochloric acid(final pH 2-3). The acid solution is dialyzed overnight against 100volumes of 0.9 sodium chloride and further dialyzed against 100 volumesphosphate buffered saline (ph 7) to give about ml. of solutioncontaining highly concentrated (about 100 fold) interferon.

EXAMPLE l0 Concentration of Interferon from Tissue Culture Fluids (frommonkey kidney cell culture) To 500 ml. of crude harvest fluid containinginterferon obtained by the method described by Hsiung, Proc. Soc. Exper.Biol. and Med, 108: 357 (1962). by inoculating monkey kidney cellcultures with heat inactivated DA myxovirus is added 26 ml. oftrichloracetic acid. The mixture is stirred about A hour at 5 C. andthen centrifuged for /2 hour at 1500 r.p.m. in a refrigeratedcentrifuge. The supernate is separated and adjusted to pH 7 with 10%sodium hydroxide and 0.2 M zinc acetate is added to a finalconcentration of about 30 millimolar. The pH again is adjusted to 7-7.5by addition of 5% sodium hydroxide and the mixture allowed to standovernight at 5 C. The precipitate is collected by centrifugation anddissolved in 5 ml. of cold (0-5 C.) hydrochloric acid (final pH 2-3).The acid solution is dialyzed overnight against 100 volumes of 0.9%sodium chloride and further dialyzed against 100 volumes phosphatebuffered saline (pH 7) to give about 5 ml. of solution containing highlyconcentrated (about 100 fold) interferon.

EXAMPLE '1 1 Concentration of Interferon from Tissue Culture Fluids(from chick embryo cell culture) To 500 ml. of crude harvest fluidcontaining interferon obtained by the method described by Lennette etal., J. Exp. Med., 83: 195 (1946) by inoculating yellow fever virus, 17DD High Strain, into chick embryo cell cultures is added 50 ml. oftrichloracetic acid. The mixture is stirred about /2 hour at 5 C. andthen centrifuged for /2 hour at 1500 r.p.m. in a refrigeratedcentrifuge. The supernate is separated and adjusted to pH 7 with 10%sodium hydroxide and 0.2 M zinc acetate is added to a finalconcentration of about 5 millimolar. The pH again is adjusted to 7-7.5by addition of 5% sodium hydroxide and the mixture allowed to standovernight at 5 C. The

precipitate is collected by centrifugation and dissolved in 5 ml. ofcold (0-5, C.) hydrochloric acid (final pH 2-3). The acid solution isdialyzed overnight against 100 volumes of 0.9% sodium chloride andfurther dialyzed against 100 volumes phosphate buffered saline (pH 7) togive about 5 ml. of solution containing. highly concentrated (about 50fold) interferon.

While the invention has been described by the use of certain specificconcentrating agents and certain specific conditions for furtherpurifying interferons the invention includes the use of equivalentagents and conditions falling within the scope of the appended claims.

What is claimed is:

1. A process for concentrating interferons from a fluid medium intowhich interferon has been elaborated by cells in contact with interferoninducing agents comprising adding to said fluid medium maintained at atemperature between 0 to 30 C. a protein precipitating agent selectedfrom the group consisting of perchloric acid, trichloracetic acid,sulfosalicylic acid and tetrametaphosphate to a final concentration of13% of said interferon-containing fluid, adjusting the supernate to pH6-8 and then adding a heavy metal salt selected from the groupconsisting of cobalt, zinc, calcium, mercury, strontium and barium saltsof acetic acid, glycine, lactic acid,

propionic acid and hydrochloric acid to coprecipitate the interferon. I

2. A process as claimed in claim 1, wherein the interferon is separatedfrom the heavy metal salt coprecipitan by adjusting the pH to betweenabout 2 to 3.

3. A process as claimed in claim 1, wherein the interferon is separatedfrom the heavy metal salt coprecipitant by addition ofethylenediaminetetraacetic acid tetrasodium salt.

4. A process for concentrating interferons from a fluid medium intowhich interferon has been elaborated by cells in contact withinterferoninducing agents comprising adding to said fluid medium maintained at atemperature between 0 to 30 C., perchloric acid to a final concentrationof between 1-13% of said interferon-containing fluid, adjusting thesupernate to between pH 6-8 and then adding zinc acetate tocoprecipitate the interferon.

5. A process as claimed in claim 4, wherein the precipitate containingzinc acetate and interferon is taken up into solution at pH betweenabout 2 to 3 and then dialyzed to remove the zinc acetate.

6. A process for concentrating an interferon prepared by injecting,allantoically, an inoculum dose of influenza virus into 9 to 12 dayoldembryonated avian eggs, incubating at least 72 hours at 3637 C.,chilling the eggs and withdrawing the allantoic fluid containing theinduced interferon, comprising adding to said fluid at a temperaturemaintained between 0 to 30 C. a protein precipitating agent selectedfrom the group consisting of perchloric acid, trichloracetic acid,sulfosalicylic acid and tetrametaphosphate to a final concentration ofl-3% of the interferon-containing allantoic fluid, adjusting thesupernate to pH 6-8 and then adding a heavy metal salt selected from thegroup consisting of cobalt, zinc, calcium, mercury, strontium andbariumsalts of acetic acid, glycine, lactic acid, propi-onic acid andhydrochloric acid to coprecipitate the interferon,

7. A process for concentrating interferon prepared by injecting,allantoically, an inoculum dose of influenza virus into 9 to 12 day-oldembryonated avian eggs, incubating at least 72 hours at 36-37 C.,chilling the eggs and withdrawing the allantoic fluid containinginterferon, comprising adding to the interferon-containing allantoicfluid, maintained at a temperature between 0 to 30 C. perchloric acid toa final concentration of between about 1-3%, adjusting the supernate tobetween about pH 6-8 and then adding zinc acetate to coprecipitate theinterferon.

8. A process as claimed in claim 7, wherein the precipitate containingzinc acetate and interferon is taken up in solution at about pH 2-3 andthen dialyzed to remove zinc acetate.

9. A process for concentrating interferon from tissue culture fluid intowhich interferon has been elaborated by cells in contact with interferoninducing agents, comprising adding to said fluid maintained at atemperature between 0 to 30 C. a protein precipitating agent selectedfrom the group consisting of perchloric acid, trichloracetic acid,sulfosalicylic acid and tetrametaphosphate to a final concentration of1-3% of said interferon-containing fluid, adjusting the supernate to pH6-8 and then adding a heavy metal salt selected from the groupconsisting of cobalt, zinc, calcium, mercury, strontium and barium saltsof acetic acid, glycine, lactic acid, propionic acid and hydrochloricacid to coprecipitate the interferon.

10. A process as claimed in claim 9, wherein perchloric acid is employedas the protein precipitating agent, and zinc acetate is employed tocoprecipitate the interferon.

11. A process as claimed in claim 10, wherein the precipitate containingZinc acetate and interferon is taken up in solution at about pH 2-3 andthen dialyzed to remove the zinc acetate component.

12. A process as claimed in claim 1, comprising taking the coprecipitateof heavy metal salt and interferon into solution at about pH 2-3,dialyzing the coprecipitate to remove the heavy metal salt, bufieringthe interferoncontaining solution to between about pH 5.86. 'r and anionic strength between about 003-008, adding the solution to acarboxylic cation exchange resin adsorbent equilibrated to between aboutpH 5.8-6.4, removing inactive material by passing over the resinadsorbent a buffer having between about pH 5.86.4, a stepwise increasein ionic strength from between about 0.03-0.08 to about 0.13 and thendesorbing interferonby passing over the resin adsorbent a buffer havingbetween about pH 7.4-8.8 and an ionic strength of between about 0.13-0.18.

13. A process as claimed in claim 12, wherein the elution of inactiveand active material from the carboxylic cation exchange resin adsorbentis done by a stepwise ionic strength and pH gradient in a bufferselected from the group consisting of acetate and phosphate buffer.

14. A process as claimed in claim 12, wherein the interferon componentin the fluid obtained is separated from the major portion of residualimpurities by Zone electroporesis at temperatures below roomtemperature.

15. A process for concentrating and purifying interferon prepared byinjecting, allantoically, an inoculum dose of influenza virus into 9 to12 day-old embryonated avian eggs, incubating at least 72 hours at 36-37C., chilling the eggs and withdrawing the allantoic fluid containinginterferon, comprising adding to said interferon- 20 containing fluidmaintained at a temperature betfeen 0 to C., perchloric acid to a finalconcentration of between about 13%, adjusting the supernate to betweenabout pH 68 and'then adding zinc acetate to coprecipitate interferon,taking the precipitate into solution at pH between about 2 and 3 andthen dialyzing the interferon against a buffer having a pH of about 6and an ionic strength of between about 0.030.08 to remove the zincacetate, adding a phosphate buffer pH 6 to the interferoncontainingsolution, adding the solution to a carboxymethylcellulose columnequilibrated to about pH 6, removing inactive substance by stepwisepassing over the column phospate buffer, pH 6, with ionic strength of0.03, then phosphate buffer, pH 6, with ionic strength of 0.08, thenphosphate bulTer, pH 6, with ionic strength 0.13 and then eluting theinterferon with phosphate buffer, pH 8, and ionic strength of 0.13.

16. A process as claimed in claim 15, wherein the interferon componentin the fluid obtained is separated from the major portion of residualimpurities by zone electrophoresis on a copolymer of polyvinylacetateand polyvinyl chloride at a temperature below room temperature andemploying from about 5 to about 10 volts per cm. length of bed.

No references cited.

LEVIS GOTTS, Primary Examiner.

1. A PROCESS FR CONCENTRATING INTERFERONS FROM A FLUID MEDIUM INTO WHICHINTERFERON HAS BEEN ELABORATED BY CELLS IN CONTACT WITH INTERFERONINDUCING AGENTS COMPRISING ADDING TO SAID FLUID MEDIUM MAINTAINED AT ATEMPERATURE BETWEEN 0 TO 30*C. A PROTEIN PRECIPITATING AGENT SELECTEDFROM THE GROUP CONSISTING OF PERCHLORIC ACID, TRICHLORACETIC ACID,SULFOSALICYLIC ACID AND TETRAMETAPHOSPHATE TO A FINAL CONCENTRATION OF1-3% OF SAID INTERFERON-CONTAINING FLUID, ADJUSTING THE SUPERNATE TO PH6-8 AND THEN ADDING A HEAVY METAL SALT SELECTED FROM THE GROUPCONSISTING OF COBALT, ZINC, CALCIUM, MERCUTY, STRONTIUM AND BARIUM SALTSOF ACETIC ACID, GLYCINE, LACTIC ACID, PROPIONIC ACID AND HYDROCHLORICACID TO COPRECIPITATE THE INTERFERON.